Neurotrophic factors are defined as secretory proteins that regulate neuronal survival and differentiation. Recent studies indicate that a major function of neurotrophic factors in the brain is to regulate synaptic transmission and plasticity. This laboratory was among the first to reveal this novel function. We demonstrated, using rodent models, that brain derived neurotrophic factor (BDNF) plays a key role in hippocampal long-term potentiation (LTP), a cellular model for learning and memory. Using the Xenopus neuromuscular synapse as a model, we identified two modes of regulation by neurotrophins: acute modulation of synaptic transmission and plasticity, and long-term alteration of the structure and function of synapses. In the period covered by this report, we have made two significant discoveries: 1) BDNF-induced translocation of TrkB into lipid rafts: functional role in synaptic transmission. BDNF plays a key role in synapse development and plasticity but the underlying signaling mechanisms remain largely unknown. We found that BDNF rapidly recruits full-length TrkB (TrkB-FL) receptor into lipid rafts, cholesterol- and sphingolipid-enriched membrane microdomains hypothesized as signaling platforms for extracellular stimuli, from non-raft regions of neuronal plasma membranes. Truncated TrkB lacking the intracellular kinase domain was not translocated, and the translocation of TrkB-FL was blocked by Trk inhibitors, suggesting that phosphorylation by TrkB tyrosine kinase is required for the translocation. Disruption of lipid rafts by depleting cholesterol from cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic response to tetanus in hippocampal slices. In contrast, lipid rafts are not required for BDNF regulation of neuronal survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent a signaling mechanism selective for synaptic modulation by BDNF in the CNS 2) Cyclic AMP controls BDNF-induced TrkB phosphorylation and dendritic spine formation in hippocampal neurons. We next examined how BDNF/TrkB signalings are controlled by cAMP. It has been hypothesized that the synaptic actions of BDNF are ?gated? by cAMP, but the underlying molecular mechanisms remain unclear. We show that cAMP regulates BDNF function by modulating TrkB receptor signaling and trafficking. TrkB phosphorylation is controlled by cAMP, with three features characteristic for cAMP gating: BDNF-induced TrkB phosphorylation was attenuated by inhibitors of cAMP signaling, potentiated by cAMP analogs, and activation of cAMP pathway alone had no effect on TrkB phosphorylation. cAMP also facilitated trafficking of TrkB to dendritic spines, possibly by promoting its interaction with synaptic scaffolding protein PSD-95. Long-term modulation of spine density, but not dendritic branching, by BDNF in cultured hippocampal neurons was gated by cAMP. These results reveal a specific role of cAMP in controlling BDNF actions in the brain, and provide new insights into the molecular mechanism underlying cAMP gating. 3) Hippocampal long-term depression (LTD) regulated by proBDNF/p75 NTR signaling. Pro- and mature brain-derived neurotrophic factor activates two distinct receptors: p75 neurotrophin receptor (p75NTR) and TrkB. Mature BDNF facilitates hippocampal synaptic potentiation through TrkB. We have now demonstrated that proBDNF, by activating p75NTR, facilitates hippocampal long-term depression (LTD). Electron microscopy localized p75NTR in dendritic spines, in addition to afferent terminals, of CA1 neurons. Genetic deletion of p75NTR in mice selectively impaired the NMDA receptor-dependent LTD, without affecting other forms of synaptic plasticity. p75NTR ?/? mutant mice also exhibited a decrease in the expression of NR2B, an NMDA receptor subunit uniquely involved in LTD. Activation of p75NTR by proBDNF enhanced NR2B-dependent LTD and NR2B-mediated synaptic currents. These results demonstrate a critical role for proBDNF-p75NTR signaling in LTD and its potential mechanism, and together with the finding that mature BDNF promotes synaptic potentiation, suggest a bi-directional regulation of synaptic plasticity by pro- and mature BDNF. 4) Interaction of the sorting motif on mature BDNF and the sorting receptor CPE required for activity-dependent secretion of BDNF. Although activity-dependent secretion of BDNF is thought to be an important mechanism that mediates synaptic plasticity and short-term memory, it is unclear how BDNF achieves regulated secretion. We have previously shown that the pro-domain, particularly the region that contains val66, is critical for activity-dependent secretion of BDNF. In collaboration with Peng Loh?s group at NICHD, we have now provide evidence that a sorting motif localized in the mature domain of BDNF that interacts with a sorting receptor, and such interaction is important in targeting BDNF to the regulated secretory pathway. X-ray crystal structure analysis revealed a putative sorting motif for regulated secretion, I16E18I105D106 in BDNF. Substitution mutation of the acidic residues in the motif resulted in missorting of proBDNF to the constitutive pathway in AtT20 cells. Introduction of an acidic residue to the relevant position in NGF, which is largely secreted constitutively, redirected a significant proportion of it into the regulated pathway. Modeling and binding studies indicated that the acidic residues in the BDNF sorting motif interact with two basic residues in the sorting receptor, carboxypeptidase E (CPE). 35S pulse?chase experiments showed that activity-dependent secretion of endogenous BDNF from cortical neurons was obliterated in CPE knockout mice. Thus, we have identified a mechanism whereby a specific motif I16E18I105D106 interacts with CPE to sort proBDNF into regulated pathway vesicles for activity-dependent secretion. Further studies are necessary to delineate the relationships between the pro- and mature domains in activity-dependent secretion of BDNF (Neuron).